Abstract

Background

The FDA-approved small-molecule drug ibrutinib is an effective targeted therapy for patients with chronic lymphocytic leukemia (CLL). Ibrutinib inhibits Bruton’s tyrosine kinase (BTK), a kinase involved in B cell receptor signaling. However, the potential regulation of neuroinflammatory responses in the brain by ibrutinib has not been comprehensively examined.

Methods

BV2 microglial cells were treated with ibrutinib (1 μM) or vehicle (1% DMSO), followed by lipopolysaccharide (LPS; 1 μg/ml) or PBS. RT-PCR, immunocytochemistry, and subcellular fractionation were performed to examine the effects of ibrutinib on neuroinflammatory responses. In addition, wild-type mice were sequentially injected with ibrutinib (10 mg/kg, i.p.) or vehicle (10% DMSO, i.p.), followed by LPS (10 mg/kg, i.p.) or PBS, and microglial and astrocyte activations were assessed using immunohistochemistry.

Conclusions

Our data provide insights on the mechanisms of a potential therapeutic strategy for neuroinflammation-related diseases.

Background

The human brain contains microglia, astrocytes, and neuronal cells. As resident macrophages in the central nervous system (CNS), microglia and astrocytes play vital roles in the innate immune response and serve as the frontline defense against exogenous toxic substances and proinflammatory reactions [1, 2]. In the normal brain, microglia play an important role in neuroprotection, and phagocytes remove cell debris and damaged neurons [3]. However, abnormally activated microglia and astrocytes significantly accelerate neuroinflammatory and neurotoxic responses by releasing various proinflammatory cytokines and mediators, including interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2), and nitric oxide synthase (iNOS) [4]. These neuroinflammatory responses are strongly correlated with neurodegenerative diseases such as Alzheimer’s disease (AD) and lead to synaptic degeneration, neuronal cell death, and cognitive dysfunction [5]. Therefore, regulation of the neuroinflammatory response represents a potential therapeutic strategy for neuroinflammation/neurodegeneration-related diseases.

Lipopolysaccharide (LPS) is a prominent cell wall component of gram-negative bacteria that is a strong stimulator of microglial activation [6]. LPS-induced microglial activation results in inflammatory responses that promote disease progression in models of neurodegeneration [7, 8]. LPS interacts with Toll-like receptors (TLRs) such as TLR4 on the surface of microglia [9]. This interaction activates TLR4 and downstream signaling pathways. Activated TLR4 signaling affects NF-κB and/or other transcription factors in the nucleus and triggers the release of proinflammatory cytokines [10]. Thus, modulating the LPS and TLR interaction and/or activation is a potential therapeutic strategy for preventing/treating neuroinflammation-related diseases.

Ibrutinib is an irreversible and selective small-molecule inhibitor of Bruton’s tyrosine kinase (BTK) [11] that can cross the blood-brain barrier [12]. BTK is a key regulator of B cell receptor functions and signaling and modulates cell survival and proliferation in various B cell malignancies. Anti-tumor activity of ibrutinib has been observed in vivo and in clinical studies [13]. According to several recent studies, ibrutinib has immunomodulatory action. For instance, treatment of mice with ibrutinib improved the anti-tumor immune response of infiltrating T cells [14]. Additionally, Kondo et al. showed that patients with chronic lymphocytic leukemia (CLL) who received ibrutinib exhibited significantly reduced STAT3 phosphorylation and IL-10 proinflammatory cytokine levels [15]. Ibrutinib is also a useful treatment for bone and autoimmune diseases, including rheumatoid arthritis [16]. As shown by Shinohara et al., oral administration of ibrutinib inhibits osteoclast resorption in the bone by targeting the integrin pathway [17]. However, researchers have not comprehensively investigated whether ibrutinib regulates neuroinflammatory responses in the brain.

In the present study, we examined the effects of ibrutinib on microglial and astrocytic proinflammatory responses and found that ibrutinib differentially regulates the neuroinflammatory responses in these cells. A decrease in TLR4/AKT/STAT3 signaling further suppressed proinflammatory cytokine levels as a downstream effect of ibrutinib. In addition, ibrutinib significantly suppressed LPS-induced BV2 microglial cell migration by modulating AKT signaling. Moreover, ibrutinib-injected wild-type mice exhibited significantly reduced microglial and astrocyte activation and decreased levels of the proinflammatory cytokines COX-2 and IL-1β. These data indicate that ibrutinib regulates LPS-stimulated neuroinflammatory responses in microglial cells and wild-type mice.

Methods

Cell lines and culture conditions

BV2 microglial cells (a generous gift from Dr. Kyung-Ho Suk) were maintained in high-glucose DMEM (Invitrogen, Carlsbad, CA, USA) with 5% fetal bovine serum (FBS, Invitrogen, Carlsbad, CA, USA) in a 5% CO2 incubator. Data from all in vitro experiments were analyzed in a semi-automated manner using ImageJ software, and the results were confirmed by an independent researcher who did not participate in the current experiments.

Wild-type mice

All experiments were performed in accordance with approved animal protocols and guidelines established by the Korea Brain Research Institute (IACUC-2016-0013). C57BL6/N mice were purchased from Orient-Bio Company (Gyeonggi-do, Korea). Male C57BL6/N mice (8 weeks old, 25–30 g) were housed in a pathogen-free facility with 12 h of light and dark per day at an ambient temperature of 22 °C. Data were analyzed in a semi-automated manner using ImageJ software, and the results were confirmed by an independent researcher who did not participate in the current experiments.

Immunohistochemistry

To determine whether pretreatment with ibrutinib alters LPS-induced neuroinflammation in vivo, wild-type mice were intraperitoneally (i.p.) administered ibrutinib (10 mg/kg) or vehicle (10% DMSO) daily for 3 days and subsequently injected with LPS (Sigma, Escherichia coli, 10 mg/kg, i.p.) or PBS. Three hours after the injection of LPS or PBS, the mice were perfused and fixed with 4% paraformaldehyde (PFA) solution, and the brain tissues were flash-frozen and sliced using a cryostat (35 μm thick). Each brain section was processed for immunohistochemical staining. The brain sections were rinsed with PBS and permeabilized with PBS containing 0.2% Triton X-100 and 0.5% BSA for 1 h at room temperature. The tissue sections were subsequently incubated with primary anti-Iba-1, anti-GFAP, anti-COX-2, or anti-IL-1β antibodies at 4 °C overnight. The next day, the tissue sections were washed with 0.5% BSA three times and incubated with a biotin-conjugated anti-rabbit secondary antibody (1:400, Vector Laboratories) for 1 h at room temperature. The sections were then rinsed with 0.5% BSA and incubated in an avidin-biotin complex solution (Vector Laboratories, Burlingame, CA) for 1 h at room temperature. After washing the sections three times with 0.1 M phosphate buffer (PB), the signal was detected by incubating the sections with 0.5 mg/ml 3,3′-diaminobenzidine (DAB, Sigma-Aldrich) in 0.1 M PB containing 0.003% H2O2. The sections were rinsed with 0.1 M PB and mounted on gelatin-coated slides, and images were captured under a bright-field microscope (Leica).

MTT assays

Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. BV2 microglial cells were seeded in 96-well plates and treated with various concentrations of ibrutinib (100 nM to 1 μM at lower doses and 1 μM to 50 μM at higher doses) for 24 h in the absence of FBS. The cells were then treated with 0.5 mg/ml MTT and incubated for 3 h at 37 °C in a 5% CO2 incubator. Absorbance was measured at 580 nm.

Immunocytochemistry

BV2 microglial cells were fixed with 4% paraformaldehyde for 10 min, washed with PBS three times, and then incubated with anti-CD11b and anti-IL-1β or anti-CD11b and anti-COX-2 antibodies in GDB buffer (0.1% gelatin, 0.3% Triton X-100, 16 mM sodium phosphate, pH 7.4, and 450 mM NaCl) overnight at 4 °C. The next day, the cells were washed with PBS three times and incubated with the following secondary antibodies for 1 h at room temperature: Alexa Fluor 488-conjugated anti-mouse and Alexa Fluor 555-conjugated anti-rabbit (1:200, Molecular Probes, USA). The cells were mounted in DAPI-containing solution (Vector Laboratories, CA, USA), and images were captured from a single plane using a confocal microscope (Nikon, Japan) and analyzed using ImageJ software. Samples were analyzed in a blinded manner using 6–10 individual images.

Enzyme-linked immunosorbent assay

To examine whether ibrutinib affects IL-1β levels, an enzyme-linked immunosorbent assay (ELISA) was performed. Briefly, BV2 microglial cells were treated with ibrutinib (500 nM) or vehicle (1% DMSO) for 30 min, treated with LPS (100 ng/ml) or PBS for 24 h. IL-1β ELISA was then performed using the conditioned medium. Mouse IL-1β ELISA kits (ELISA development reagents; R&D Systems, Minneapolis, MN) were used according to the manufacturer’s recommendations. Recombinant mouse IL-1β protein (R&D Systems) was used as a standard. The absorbance of the samples was measured at 450 nm using a microplate reader (BMG Labtech, Offenburg, Germany).

Western blotting

To determine whether ibrutinib affects ERK, P38, JNK, and AKT signaling, BV2 microglial cells were treated with ibrutinib (1 μM) or vehicle (1% DMSO) for 1 h, followed by LPS (1 μg/ml) or PBS for 45 min. After the final incubation, the cells were lysed with RIPA buffer containing protease and phosphatase inhibitor tablets (Roche, USA). Western blot analyses were performed as previously described [18], and images were analyzed using Fusion software or ImageJ software.

Wound-healing assay

Wound-healing assays were performed as previously described [18]. Briefly, BV2 microglial cells were seeded in 12-well plates and incubated until the cells reached 80–90% confluence. The cells were scratched with a cell scratcher (SPL, Korea) to create a wound. Images were captured at 0 h. Next, the cells were treated with ibrutinib (500 nM) or vehicle (1% DMSO) for 1 h, followed by LPS (100 ng/ml) or PBS for 23 h. Images were then captured.

Cell surface biotinylation

To measure the effects of ibrutinib on cell-surface levels of TLR4, BV2 microglial cells were treated with ibrutinib (1 μM) or vehicle (1% DMSO) for 30 min, followed by treatment with LPS (1 μg/ml) or PBS for 5.5 h. Surface proteins were then labeled with Sulfo-NHS-SS-Biotin under gentle shaking at 4 °C for 30 min, followed by the addition of quenching solution. The surface-labeled cells were lysed in lysis buffer, disrupted by sonication on ice, incubated for 30 min, and clarified by centrifugation (10,000 rpm, 10 min). The lysate was then added to immobilized NeutroAvidin TM gel and incubated for 1 h, followed by washing three times with wash buffer and incubation for 1 h in SDS-PAGE sample buffer with DTT. Surface proteins were then analyzed by immunoblotting with an antibody recognizing the N-terminus of TLR4.

Statistical analyses

All data were analyzed with GraphPad Prism 6 software using either unpaired two-tailed t tests with Welch’s correction for comparisons between two groups or one-way ANOVA for multiple comparisons. Post hoc analyses were performed with Tukey’s multiple comparison test with significance set at *p < 0.05, **p < 0.01, and ***p < 0.0001. Data are presented as the mean ± SEM.

Results

Ibrutinib does not exert toxic effects on BV2 microglial cells at concentrations up to 25 μM

To test the effects of ibrutinib on neuroinflammation, we first examined whether ibrutinib is toxic toward BV2 microglial cells. BV2 microglial cells were treated with vehicle (1% DMSO) or ibrutinib (100, 250, 500, 750, or 1000 nM) for 24 h, and MTT assays were conducted. Ibrutinib did not exhibit toxicity at lower doses (Fig. 1a, b). We also examined the effects of higher doses of ibrutinib on cell viability. For this purpose, BV2 microglial cells were treated with vehicle (1% DMSO) or ibrutinib (1, 5, 10, 25, or 50 μM) for 24 h, and MTT assays were conducted. We found that ibrutinib did not induce BV2 microglial cell toxicity at concentrations up to 25 μM (Fig. 1c).

To determine whether ibrutinib alters the LPS-induced morphology of BV2 microglial cells, cells were pretreated with ibrutinib (1 μM) or vehicle (1% DMSO) for 30 min, followed by treatment with LPS (1 μg/ml) or PBS for 5.5 h. The cells were then fixed in 4% paraformaldehyde and immunostained with an anti-CD11b antibody. In contrast to vehicle-treated cells, LPS-treated BV2 microglial cells displayed long thin processes extending from the cell body (Fig. 1d, middle panel). Pretreatment with ibrutinib followed by LPS treatment reduced the number of long thin processes extending from the cell body, and the morphology of these cells resembled that of vehicle-treated cells (Fig. 1d, lower panel).

LPS interacts with TLR4 on the surface of microglial cells to increase immune responses [19]. Thus, we hypothesized that ibrutinib may inhibit the LPS and TLR4 interaction on the cell surface and/or TLR4 activation to regulate neuroinflammatory responses. To test this hypothesis, BV2 microglial cells were pretreated with TAK-242 (TLR inhibitor, 500 nM) or vehicle (1% DMSO) for 30 min, followed by treatment with ibrutinib (1 μM) or vehicle (1% DMSO) for 30 min and subsequent treatment with LPS (1 μg/ml) or PBS for 5 h. Total RNA was isolated, and IL-1β and COX-2 mRNA levels were measured by RT-PCR. Consistent with our findings described above, ibrutinib significantly decreased LPS-induced COX-2 and IL-1β mRNA levels (Fig. 4a–c). In addition, treatment with TAK-242, ibrutinib, and LPS further decreased LPS-induced COX-2 mRNA levels compared with treatment with LPS and TAK-242 or ibrutinib and LPS (Fig. 4a–c). However, treatment with TAK-242, ibrutinib, and LPS did not significantly reduce LPS-induced IL-1β mRNA levels compared with treatment with LPS and TAK-242 or ibrutinib and LPS (Fig. 4a–c).

We then examined whether ibrutinib alters LPS-induced p-AKT signaling. To test this, BV2 microglial cells were treated with ibrutinib (1 μM) or vehicle (1% DMSO) for 1 h, followed by treatment with LPS (1 μg/ml) or PBS for 45 min, and western blotting was conducted with anti-p-AKT and anti-AKT antibodies (Fig. 5a–c). We found that ibrutinib significantly reduced the LPS-induced increases in p-AKT levels in BV2 microglial cells (Fig. 5a–c).

Next, we investigated the effects of ibrutinib on LPS-induced p-AKT levels in a longer treatment. BV2 microglial cells were pretreated with ibrutinib (1 μM) or vehicle (1% DMSO) for 5 h, followed by treatment with LPS (1 μg/ml) or PBS for 45 min, and western blotting was performed with anti-p-AKT and anti-AKT antibodies. Longer treatment with ibrutinib significantly reduced the LPS-mediated increases in p-AKT levels compared with LPS treatment (Fig. 5d–f). In addition, we measured whether ibrutinib itself affects AKT phosphorylation in the absence of LPS. For this experiment, BV2 microglial cells were treated with ibrutinib (1 μM) or vehicle (1% DMSO) for 6 h, and western blotting was performed with anti-p-AKT and anti-AKT antibodies. Unexpectedly, ibrutinib alone significantly reduced p-AKT levels in BV2 microglial cells, but the total AKT levels were unchanged (Additional file 1: Figure S5a–c).

To test whether ibrutinib modulates AKT signaling to alter LPS-mediated proinflammatory responses, BV2 microglial cells were pretreated with the MK2206 (a AKT inhibitor, 10 μM) or vehicle (1% DMSO) for 30 min, treated with ibrutinib (1 μM) or vehicle (1% DMSO) for 30 min, treated with LPS (1 μg/ml) or PBS for 5 h, and mRNA levels of COX-2 and IL-1β were measured by RT-PCR. Treatment with MK2206, ibrutinib, and LPS significantly suppressed the mRNA levels of IL-1β compared with treatment with ibrutinib and LPS or MK2206 and LPS (Fig. 5g–i). By contrast, treatment with MK2206, ibrutinib, and LPS did not significantly reduce the mRNA levels of COX-2 compared with treatment with ibrutinib and LPS or MK2206 and LPS (Fig. 5g–i). These data indicate that the effects of ibrutinib on proinflammatory responses in LPS-induced BV2 microglial cells partially depend on AKT signaling.

According to recent studies, activated microglia and astrocytes are associated with proinflammatory responses and neuroinflammation [7]. Thus, we examined the effects of ibrutinib on LPS-induced microglial and astrocyte activation in vivo. Wild-type mice were first injected with ibrutinib (10 mg/kg, i.p.) daily for 3 days and then injected with LPS (10 mg/kg, i.p.) or PBS. Three hours after injection with LPS or PBS, immunohistochemistry was conducted with anti-Iba-1 or anti-GFAP antibodies. The LPS-injected wild-type mice showed a significant increase in microglial and astrocyte activation (Fig. 9), whereas ibrutinib significantly inhibited microglial (Fig. 9a–c) and astrocyte (Fig. 9d–f) activation in the cortex and hippocampus of LPS-injected wild-type mice.

Next, we investigated whether ibrutinib regulates the LPS-stimulated increase in IL-1β and COX-2 levels in LPS-injected wild-type mice. For this experiment, wild-type mice were first injected with ibrutinib (10 mg/kg, i.p.) daily for 3 days and then injected with LPS (10 mg/kg, i.p.) or PBS. Three hours after the injection with LPS or PBS, immunohistochemistry was conducted with anti-IL-1β or anti-COX-2 antibodies. The LPS-injected wild-type mice exhibited a significant increase in IL-1β levels compared with vehicle-injected wild-type mice (Fig. 10a–e). In addition, ibrutinib were significantly decreased IL-1β levels in the cortex but not the hippocampus in LPS-injected wild-type mice (Fig. 10). Moreover, ibrutinib were significantly downregulated COX-2 levels in the hippocampus (Fig. 11a–c) and cortex (Fig. 11d, e) in LPS-injected wild-type mice. These data suggest that ibrutinib modulates LPS-induced microglial and astrocyte activation as well as the levels of the proinflammatory cytokines COX-2 and IL-1β in vivo.

Discussion

Microglia and astrocytes are the first line of defense in the central nervous system (CNS) and initiate immune responses to injuries and pathogens [24]. Activated microglia and astrocytes release a variety of proinflammatory cytokines [25, 26]. Specifically, abnormally activated microglia produce a variety of inflammatory mediators (COX-2 and iNOS) and inflammatory cytokines (IL-1β, IL-6, and TNF-α). During pathological conditions involving CNS inflammation, IL-1β is mainly released by activated macrophages and microglia, and astrocytes are regarded as the major target of IL-1β, as suggested by the presence of IL-1β receptors on the surfaces of astrocytes [27]. In astrocytes, IL-1β induces the expression of other cytokines, including IL-6 and TNF-α, as well as other inflammatory mediators that have been implicated in the CNS immune response to injury [28]. Interestingly, systemic LPS and IL-1β injections have been reported to induce excess COX-2 production within the rodent brain [29, 30]. The COX-2 expression is substantially increased in the frontal cortex and hippocampus in the brains of subjects with Alzheimer’s disease (AD) [31]. Therefore, drugs modulating microglial activation and the release of proinflammatory cytokines that effectively inhibit inflammation represent a promising therapeutic strategy for neuroinflammation/neurodegeneration-related diseases. Not surprisingly, ibrutinib itself did not alter proinflammatory cytokine levels in BV2 microglial cells compared with vehicle treatment in the present study (Fig. 2, Additional file 1: Figure S1), suggesting that ibrutinib alone does not affect the levels of any proinflammatory cytokines under basal conditions. However, ibrutinib significantly reduced proinflammatory cytokine levels in LPS-induced BV2 microglia (Fig. 2, Additional file 1: Figure S1) and primary microglia (Fig. 3) but not primary astrocytes (Additional file 1: Figure S2). In addition, pretreatment with ibrutinib reduced proinflammatory cytokine levels more effectively than post-treatment, highlighting the potential of ibrutinib as a preventive drug (Fig. 2, Additional file 1: Figure S1). Based on these findings, we speculate that pre- or post-treatment with ibrutinib differentially modulates LPS-induced proinflammatory cytokine production depending on the cell type.

The members of the TLR family are the main mediators of the innate immune response. TLRs are mainly expressed in immune cells and have also been identified in different CNS cell types, such as microglia, astrocytes, or cells in the cerebral microvasculature [32]. TLR4 is the most representative member of the TLR family and predominantly responds to LPS through its co-receptor, myeloid differentiation protein-2 (MD-2), which is essential for LPS-induced stimulation of TLR4 [33]. TLR4 binds to some other adapter proteins, including myeloid differentiation factor 88 (MyD88), to activate downstream signaling. Specifically, the interaction between LPS and TLR4 activates MARK signaling pathways (including AKT) in BV2 microglial cells [34]. Therefore, abnormal TLR4 expression or abnormal immune responses might damage the CNS. Interestingly, in the present study, treatment with TAK-242 (a TLR4 inhibitor), ibrutinib, and LPS further decreased LPS-induced COX-2 mRNA levels compared with the treatment with TAK-242 and LPS or ibrutinib and LPS (Fig. 4a, b). However, treatment with TAK-242, ibrutinib, and LPS did not reduce LPS-induced IL-1β mRNA levels compared with treatment with TAK-242 and LPS or ibrutinib and LPS (Fig. 4a–c). These data suggest that ibrutinib alters TLR4 and/or other neuroinflammation-related receptors to modulate LPS-induced proinflammatory cytokine levels.

How does ibrutinib downregulate proinflammatory cytokine levels? Ibrutinib may inhibit the interaction between LPS and TLR4 on the cell surface and thereby deactivate downstream signaling pathways to suppress the neuroinflammatory response. Interestingly, we found that ibrutinib decreased LPS-induced cell-surface levels of TLR4 compared with LPS treatment (Additional file 1: Figure S3a, b). Another possible mechanism is that ibrutinib directly or indirectly suppresses TLR4 activation to reduce neuroinflammatory responses via other neuroinflammatory-related receptors that interact with LPS. Based on our findings, ibrutinib may regulate cell-surface levels of TLR4 to inhibit the interaction between TLR4 and LPS on the cell surface to alter neuroinflammatory responses. Future studies will examine whether ibrutinib modulates the LPS and TLR4 interaction and/or other neuroinflammatory-related receptors to regulate neuroinflammation.

AKT signaling plays an important role in the LPS-induced proinflammatory response [35]. AKT is the main kinase in the signal transduction pathway predominantly responsible for the production and synthesis of proinflammatory mediators and modulates TLR4 expression [36]. For instance, AKT negatively regulates LPS-induced TNF-α and IL-6 levels in the bone marrow macrophages [37]. Phosphorylated AKT also promotes the expression of inflammatory molecules, including iNOS and COX-2 [38]. As shown by Saponaro et al., LPS binds to TLR4 and activates AKT signaling to alter the production of the proinflammatory cytokine iNOS in microglial cells [39]. Thus, the maintenance of a homeostatic balance in AKT signaling might play an important role in its anti-inflammatory effects. In the present study, we found that ibrutinib dramatically reduced LPS-induced AKT phosphorylation in BV2 microglial cells (Fig. 5). Unexpectedly, we observed that ibrutinib further decreased LPS-induced AKT phosphorylation compared with vehicle to below basal levels (ibrutinib+LPS vs vehicle, Fig. 5). We therefore tested whether ibrutinib itself alters p-AKT levels and found that ibrutinib alone significantly reduced p-AKT levels compared with vehicle treatment (Additional file 1: Figure S5). Since ibrutinib alone decreased p-AKT levels, we investigated whether ibrutinib itself regulates proinflammatory cytokine levels and found that ibrutinib alone did not reduce any proinflammatory cytokine levels compared with vehicle treatment (Fig. 2, Additional file 1: Figure S1). However, AKT inhibition selectively regulated LPS-induced proinflammatory cytokine levels in the presence of ibrutinib (Fig. 5g–i). Based on our findings and the literature, we suggest that ibrutinib inhibits AKT phosphorylation to alter LPS-induced neuroinflammatory responses. In addition, ibrutinib itself may affect anti-inflammatory cytokine levels to regulate neuroinflammatory responses or may affect another biological function (i.e., phagocytosis) in the presence/absence of LPS in BV2 microglial cells. Future studies will explore whether ibrutinib itself modulates anti-inflammatory effects and how ibrutinib regulates p-AKT levels in the absence of LPS, as well as the molecular mechanisms by which ibrutinib differentially regulates neuroinflammatory responses and/or other biological functions in the absence/presence of LPS in microglial cells.

Systemic injections of LPS promote microglial and astrocyte activation and increase proinflammatory cytokine levels in wild-type mice [51]. Skelly et al. found that even a single injection of LPS induces a robust expression of the proinflammatory cytokines IL-1β and COX-2 in the hippocampus in wild-type mice [52]. LPS induces neuroinflammation in the mouse brain, as evidenced by increased immunostaining for Iba-1 (for microglial cells) and GFAP (for astrocytes) [53]. In the present study, LPS-injected wild-type mice exhibited significantly increased microglial and astrocyte activation. Ibrutinib strongly inhibited this LPS-mediated microglial and astrocyte activation (Fig. 9) as well as the increase in COX-2 and IL-1β proinflammatory cytokine levels (Figs. 10 and 11), suggesting that ibrutinib has potential as a targeted drug for neuroinflammation-related diseases.

Acknowledgements

Confocal microscopy (Nikon, TI-RCP) data were acquired at the Advanced Neural Imaging Center at the Korea Brain Research Institute (KBRI).

Funding

This work was supported by grants from the KBRI Basic Research Program through the Korea Brain Research Institute funded by the Ministry of Science, ICT and Future Planning (grant number 18-BR-02-04, HSH; 18-BR-03-02, JK) and the National Research Foundation of the Korean government (grant number 2016R1A2B4011393, HSH).

Availability of data and materials

All data generated and/or analyzed during this study are included in this article.

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Contributions

HSH conceived the study and participated in the design of the study and writing of the manuscript. HYN, JHN, HJK, JYL, and HJC performed the molecular/cellular experiments, in vivo experiments, and statistical analyses. JYL, GY, YN, and JK performed the microglial cell migration assays and functional assays, confirmed all the data analyses from all figures, and participated in the writing and editing of the revised manuscript. All authors read and approved the final manuscript.

Corresponding author

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Authors’ information

All authors are in the Neurodegenerative Disease Laboratory, Department of Neural Development and Disease, Korea Brain Research Institute, Daegu, Korea.

Ethics approval and consent to participate

All animal experiments were performed in accordance with the approved animal protocols and guidelines established by the Korea Brain Research Institute Animal Care and Use Committee (IACUC-2016-0013). Consent to participate for human subjects is not applicable in this study.

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The authors declare that they have no competing interests.

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